INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY

ISSN Print: 1560–8530; ISSN Online: 1814–9596

13–1198/2014/16–3–564–570

http://www.fspublishers.org

Full Length Article

To cite this paper: Roohi, A., I. Ahmed, N. Khalid, M. Iqbal and M. Jamil, 2014. Isolation and phylogenetic identification of halotolerant/halophilic bacteria

from the salt mines of Karak, Pakistan, Int. J. Agric. Biol., 16: 564‒570

Isolation and Phylogenetic Identification of Halotolerant/Halophilic

Bacteria from the Salt Mines of Karak, Pakistan

Aneela Roohi1,2

, Iftikhar Ahmed1*

, Nauman Khalid3, Muhammad Iqbal

1 and Muhammad Jamil

2*

1National Institute for Genomics and Advanced Biotechnology (NIGAB), National Agricultural Research Centre

(NARC), Park Road, Islamabad-45500, Pakistan 2Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Pakistan

3Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku 113-8657, Tokyo, Japan

*For correspondences: iftikharnarc@hotmail.com; jamilkhattak@yahoo.com

Abstract

Extreme environments like salt mines are inhabited by a variety of bacteria that are well-adapted to such environments. The

bacterial populations provide economic benefits in terms of enzymes synthesis. The salt mines of Karak region in Pakistan are

extremely saline and the microbial communities found here have not yet been explored. In the present study, 57

halotolerant/halophilic bacterial strains were isolated from the salt mines of Karak. These strains were grown in media with 0-

35% NaCl concentration. The morphological and physiological characteristics of the isolated strains were studied to optimize

the growth conditions and to classify the isolated bacterial strains into slightly halotolerant/halophilic, moderately halophilic

and extreme halophilic. The phylogenetic analyses inferred from 16S rRNA gene sequence of the isolated strains

demonstrated that the major population were closely related to species belonging to Planococcus, Jeotgalicoccus,

Staphylococcus, Halobacillus, Halomonas, Brevibacterium, Gracilibacillus, Kocuria, Salinivibrio, Salinicoccus,

Oceanobacillus and Bacillus genera. Results showed that the salt mines of Karak region are rich in halotolerant/halophilic

bacterial population with diverse bacterial communities, which may be utilized in various industrial applications after proper

screening and identification. © 2014 Friends Science Publishers

Keywords: Extremophile; Halotolerant; Halophilic; Karak region; 16S rRNA gene sequence

Introduction

Bacteria play an important role in the development of viable

habitats. Saline environments harbor taxonomically diverse

group of bacteria that show improved structural and

physiological characteristics in extreme (saline) conditions.

To survive in saline environments, bacteria developed

adoptive mechanisms like surface layer modifications

especially in cellular phospholipid composition (Ventosa et

al., 2008). An increasing interest in microorganisms from

hyper-saline environments has resulted in the discovery of

several new species and genera in Bacteria and Archaea

domains (Woese and Fox, 1977). Extremely halophilic

Archaea belong to the order Halobacteriales, which contains

one family, the Halobacteriaceae (Ozcan et al., 2007). The

family Halobacteriaceae belongs to domain Archaea and

presently comprised of 40 genera (Euzéby, 2013).

Among the halophilic microorganisms, Archaea are

the most common (Antón et al., 2000; Oren, 2002).

Microorganisms that form part of domain Archaea require

high salt concentration for growth and are coined as

halobacteria (Kamekura, 1998). These groups of microbes

have been isolated from diverse environments like salterns,

salt mines, salted food, saline lakes, salt marshes and saline

desert soils (Ventosa et al., 2008). Halobacteria are either

Gram-negative or Gram-positive and have aerobic,

facultative anaerobic or obligatory anaerobic metabolism

(Johnson et al., 2007). Halophilic bacteria have the ability to

grow in media containing 3 to 15% (w/v) NaCl (Oren and

Rodrı́guez-Valera, 2001; Oren, 2002). Halophilic

microorganisms have many biotechnological applications

like β-carotene production from fermented foods, hydrolytic

enzymes or exopolysaccharides, stabilizers and other

valuable compounds (Quesada et al., 1982, 2004;Ventosa et

al., 2008).

Bahadur Kheil and Jatta Ismail Kheil are two of the

biggest and well protected salt mines in Karak District,

Khyberpakhtun Khwa (KPK) Province of Pakistan, and it

has an estimated salt reserve of about 52,563 tons (PMDC,

2013). Being of high quality, Karak rock salt is consumed as

table salt after a simple treatment instead of complex

refining. However, there are insufficient reports on

exploration of bacterial diversity with respect to halophilic/

halotolerant characteristics from Pakistan (Roohi et al.,

2012). Exploration of such diversity might result in

considerable economic benefits in food and leather

Diversity of Halotolerant/Halophilic Bacteria / Int. J. Agric. Biol., Vol. 16, No. 3, 2014

565

industries. The objective of this research was to explore the

novelty associated with extremely halotolerant/halophilic

aerobic or facultative anaerobic bacteria and to assess the

bacterial biodiversity of these extremophiles from Pakistan

using molecular techniques that has not been previously

reported.

Materials and Methods

Collection of Samples

The samples were collected from the Karak Salt Mines of

KPK Province, Pakistan. The area is situated at 32° 47 to

33° 28 North and 70° 30 to 71° 30 East. The sampling sites

were divided into two different areas according to the

difference in location of salt quarries, which are located at

Bahadur Khel and Jatta Ismail Khel. Different salt mines

were selected for the sampling (Fig. 1). The salt beds are

approximately 105 m thick in Bahadur Khel, while more

than 30 m thick in Jatta Ismail Khel. Different rock salt was

collected from 5 rock salt mines. The samples were

randomly taken in sterile plastic bags and vials. These

samples were transported to the lab for the isolation of

bacteria and processing for further analysis.

Physicochemical Analysis of Brine Samples

Physico-chemical properties of the brine samples taken

from the salt mines (Bahadur Khel and Jatta Ismail Khel)

were determined including moisture contents, water

insoluble impurities SO42–

, Cl–, HCO3

–, CO3

–2 with standard

procedures while Na+, K

+, Ca

2+, Mg

2+, and some trace

elements like Zn2+

, Cu2+

, Fe2+

, Mn2+

, Pb2+

, Cr2+

and Cd2+

were determined with atomic absorption spectrophotometer

(Z-8000, Hitachi, Japan). The pH meter (Model, 3510

Jenway) was employed for pH measurement, while

laboratory thermometer was used to determine temperature

on site.

Isolation and Enrichment of Halophilic Bacteria

To recover moderately to extremely halotolerant/halophilic

bacteria, enrichment cultures and isolation procedures were

performed by dilution plate method on tryptic soy agar

medium. 1-2 mL of diluted sample was streaked on agar

medium containing various concentrations (5-25%, w/v) of

NaCl and incubated at 28ºC. The isolated strains were sub-

cultured several times under same conditions to obtain pure

cultures of morphologically different bacteria. The purified

strains were stored at – 80ºC for further characterization.

Characterization and Identification of Halotolerant

Bacterial Strains

The purified bacterial colonies were morphologically

characterized for colony color, form, elevation, margin etc.

Bacterial strains were characterized on the basis of Gram’s

staining, cell morphology and motility using microscope

(Olympus, CX31 equipped with Digital Camera 5A). The

growth characteristics of bacterial strains were determined

at various pH range (4-11), NaCl concentrations (0-40%)

and temperatures (4, 10, 20, 28, 35, 37, 40, 45 and 50ºC).

PCR Amplification and Sequencing of 16S rRNA Gene

Genomic DNA extraction for amplification of 16S rRNA

gene was performed as described by Roohi et al. (2012)

by suspending few isolated colonies in TE buffer in a

micro-centrifuge tube. These cells were heated for 10 min at

95ºC and were centrifuged at 6,000 rpm for 5 min.

Table 1: Chemical analysis of rock salt samples collected

from different location from Bahadur Khel and Jatta Ismail

Khel salt mines

Element (%)

Sulfur (SO3-2) 1.31

Calcium (CaO) 1.73

Potassium (K+) 1.65

Magnesium (Mg+2) 0.11

Chloride (Cl-1) 53.91

Sodium (Na+) 34.70

Moisture 0.13

Water insoluble impurities 7.98

Trace elements (mg/Kg)

Zinc (Zn) 0.15

Copper (Cu) 0.02

Iron (Fe) 0.57

Manganese (Mn) 0.00

Lead (Pb) 0.02

Chromium (Cr) 0.37

Cadmium (Cd) 0.00

Fig. 1: Location of sample collection sites in Karak Salt

Mines, Khyber Pakhtunkhwa Province, Pakistan

Roohi et al., / Int. J. Agric. Biol., Vol. 16, No. 3, 2014

566

The supernatant was used as template DNA for the

amplification of 16S rRNA gene. The 16S rRNA gene of all

the isolated strains was amplified by polymerase chain

reaction using primers 9F (5'-

GAGTTTGATCCTGGCTCAG-3') and 1510R (5'-

GGCTACCTTGTTACGA-3') using Premix ExTaq

(Takara, Japan) following protocol described previously

(Ahmed et al., 2007). The PCR was carried out in ABI

Veriti PCR Machine (Applied Biosystems, USA) using

optimized PCR Program: initial denaturation at 94ºC for 2

min; 30 cycles of denaturation at 94ºC for 1 min, annealing

at 50ºC for 1 min and extension at 72ºC for 1:30 min. The

final extension was performed at 72ºC for 5 min. The

amplified PCR products of 16S rRNA gene of bacterial

strains were purified and sequenced using the primers 27F

(5' AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-

ACCTTGTTACGACTT-3') using commercial service of

Macrogen Inc. Korea (http://dna.macrogen.com/eng/).

Phylogenetic Analysis of Bacterial Isolates

BioEdit software (Hall, 1999) was used to assemble the

fragment sequences of 16S rRNA gene. The 16S rRNA

gene sequences were submitted to DDBJ

(http://www.ddbj.nig.ac.jp). Using 16S rRNA gene

sequences, the strains were identified by BLAST search on

EzBiocloud Server). Closely related type species sequences

were retrieved to assess the molecular evolutionary relations

and to construct phylogenetic tree using MEGA version 5

(Tamura et al., 2011). A phylogenetic tree was constructed

from unambiguously aligned nucleotides using the

neighbour-joining (NJ) algorithm. The relationship stability

was evaluated by boot strap analysis performed using 1000

re-samplings of the neighbour-joining data for the tree

topology.

Results

Physical and Chemical Properties of Samples

The rock salt samples named M1, M2, M3, M4 and M5 were

collected from 5 different sites. The results indicated that the

mineral content, pH and temperature of samples were

suitable for the growth of halophilic bacteria. The average

temperature of all samples ranged from 23.8 to 34.7°C. The

above temperature varied according to sampling sites and

changes in air temperature. The chemical analysis showed

that major components in samples were Cl- (53.91%) and

Na+ (34.70%). Small amount of Mg

2+ (0.11%) and trace

elements were also detected from rock salt. Cd2+

was absent

from observed salt samples (Table 1). The average pH of

samples ranged between 7.8 to 7.95.

Colony, Cell Morphology and Density of

Halotolerant/Halophilic Bacteria

Fifty seven strains were isolated and purified on the basis

of morphology. Pigmentation of the strains included pale

yellow, creamy and whitish colonies. Most of the colonies

had entire margin, while some had undulate and wavy

filamentous margins (Table 2). Few of them also had lobate

margins. The growth conditions of all the isolated strains

were optimized for pH, NaCl tolerance and temperature.

Our results showed that the bacterial strains grew best at pH

7 to 9 and at a temperature range of 28-37oC. The isolated

strains toleratedNaCl up to 0-30% and based on

optimization conditions the isolated strains were classified

as slightly halotolerant (0-5% NaCl), moderately halophilic

(5-15% NaCl) and extremely halophilic (15-35% NaCl).

Phylogenetic Analysis

Phylogenetic analysis of the halophilic isolated strains

were performed by constructing a phylogenetic tree based

on the 16S rRNA gene sequences (Fig. 2). Phylogenetic

analysis indicated that the majority of isolated strains

belonged to the genera, Planococcus, Jeotgalicoccus,

Staphylococcus, Thalassobacillus, Halobacillus,

Halomonas, Brevibacterium, Gracilibacillus, Kocuria,

Salinivibrio, Salinicoccus ,Oceanobacillus and Bacillus. All

strains shared more than 97% identity with their closest

phylogenetic relatives except the strain NCCP-176. The

phylogenetic analyses showed that eight strains (NCCP-71,

91, 93, 168, 176, 701, 705, 710) can further be studied

taxonomically to delineate as novel species. The

phylogenetic analysis reflected the evolutionary

relationships among halophilic bacteria (Table 3).

Discussion

In the present study, we isolated and characterized

extremely halotolerant/halophilic aerobic or facultative

anaerobic bacteria from Pakistan using molecular

techniques that has not been previously reported. The above

growth condition are in line with several other studies that

concluded that most species show optimum growth at 3.5 to

4.5 M NaCl and pH 7.0 to 7.5 (Kushner, 1985; Oren and

Rodrı́guez-Valera, 2001). Similarly, our results of chemical

analysis of rock salt showed that these contain proper

amounts of essential ions to support the growth of extremely

halophilic microorganisms (Mancinelli and Hochstein,

1986; Oren, 1993; Birbir et al., 2007).

The colonial morphology of the isolated strains were

described using standard microbiological criteria. These

standards are based on colonial pigmentation, opacity,

diameter, consistency and elevation (Oren et al., 1997). In

these studies, we observed polymorphism with respect to

colonial morphology of the isolated strains as reported by

Castillo et al. (2007). Halobacterial members exhibited

various tones pigmentation ranging from red to pink-orange

due to high rate of carotenoid pigments in their cell

membrane. In addition, appeared opaque, transparent or

translucent, mucoid or non mucoid, with entire edges and

convex (Oren et al., 1997; Castillo et al., 2006). The

bacteria isolated from the various samples belonged to

Diversity of Halotolerant/Halophilic Bacteria / Int. J. Agric. Biol., Vol. 16, No. 3, 2014

567

diverse group of halotolerant and halophilic bacteria with

different phenotypic characteristics. However, the

phenotypic characteristics alone were not enough to

differentiate the bacterial isolates and could lead to false

identification. The main reason for this is that the

phenotypic characteristics depend on the growth conditions,

which were different in terms of NaCl concentration,

temperature, pH and medium composition. In this regard,

Fritze (2002) recommended that phenotypic characterization

results cannot be directly compared without full background

knowledge of the accurate conditions used for a particular

test.

Several studies have assessed the taxonomic status,

ecological characteristics, phylogenetic relationship and

biotechnological applications of halobacteria (Litchfield and

Gillevet, 2002). Halophilic bacteria were categorized on the

Table 2: Phenotypic characteristics of isolated halotolerant/halophilic strains

Strain ID Gram

stain

Cell

shape

Colony

color

Form Surface Margin Opacity Motility A/An pH for range

growth

(Optimum)

NaCl Range

(%) for

growth

Temperature Range

(°C) for growth

(Optimum)

NCCP-59 + R C Irregular Rough Lobate Opaque + A 5-9 (7) 2-20 4-45 (28)

NCCP-60 + C O Round Smooth Entire Opaque + A 6-9 (8) 1-15 4-40 (30)

NCCP-61 + R PY Round Smooth Entire Opaque + A 5-9 (7) 2-25 10-45 (35)

NCCP-62 + R PY Irregular Smooth Lobate Opaque + An 6-9 (8) 3-20 4-50 (30)

NCCP-63 + R PY Irregular Smooth Lobate Opaque + A 6-8 (7) 2-20 10-40 (35)

NCCP-65 + R W Irregular Rough Undulate Opaque + A 5-9 (7) 1-20 10-50 (35)

NCCP-66 + R PY Circular Shiny Entire Opaque - An 6-9 (8) 1-30 4-40 (30) NCCP-71 + R C Irregular Smooth Filamentous Opaque + A 5-10 (7) 1-25 4-50 (37)

NCCP-73 + R W Irregular Rough Undulate Opaque + A 5-9 (7) 1-20 10-50 (35)

NCCP-74 + R W Irregular Rough Undulate Opaque + A 5-9 (7) 1-20 10-50 (35)

NCCP-75 + R W Irregular Rough Undulate Opaque + A 5-9 (7) 1-20 10-50 (35)

NCCP-77 + R PY Round Smooth Entire Opaque + A 5-9 (7) 2-25 10-45 (35)

NCCP-79 + R C Round Smooth Entire Opaque + A 5-9 (8) 2-15 10-40 (28)

NCCP-80 + R PY Round Smooth Entire Opaque + A 5-9 (7) 2-25 10-45 (35)

NCCP-81 + R C Round Smooth Entire Opaque + A 5-9 (8) 0-15 10-40 (35) NCCP-82 + R C Irregular Smooth Filamentous Opaque + A 5-10 (7) 1-25 4-50 (37)

NCCP-83 + R W Irregular Rough Undulate Opaque + A 5-9 (7) 1-20 10-50 (35)

NCCP-84 + C C Round Smooth Entire Opaque - A 5-9 (8) 2-30 10-40 (35)

NCCP-85 + R W Irregular Rough Undulate Opaque + A 5-9 (7) 1-20 10-50 (35)

NCCP-86 + R PY Circular Smooth Entire Opaque + A 6-9 (7) 2-15 4-50 (28)

NCCP-87 - R C Circular Smooth Entire Opaque + A 6-8 (7) 0-15 4-45 (35)

NCCP-88 + R W Irregular Rough Undulate Opaque + A 5-9 (7) 1-20 10-50 (35) NCCP-91 + R PY Irregular Smooth Lobate Opaque + An 5-9 (7) 3-20 4-50 (30)

NCCP-92 + R PY Round Shiny Entire Opaque - A 6-9 (7) 2-20 10-45 (35)

NCCP-93 + R W Round Mucoid Entire Transparent - A 6-10 (7) 2-20 10-45 (30)

NCCP-166 + R C Irregular Smooth Filamentous Opaque + A 5-10 (7) 1-25 4-50 (37)

NCCP-168 + R PY Circular smooth Entire Opaque - A 5-9 (8) 0-15 10-40 (35)

NCCP-170 + R C Circular Smooth Filamentous Opaque + A 6-9 (7) 2-25 4-40 (37)

NCCP-171 - R W Irregular Smooth Lobate Opaque + A 5-9 (7) 1-19 10-45 (37)

NCCP-172 + R C Circular Rough Irregular Opaque + A 5-10 (7) 1-25 4-50 (37) NCCP-173 + R C Circular Rough Irregular Opaque + A 6-8 (7) 2-20 10-40 (35)

NCCP-174 + R C Round Smooth Entire Opaque + A 5-9 (8) 0-15 10-40 (35)

NCCP-175 + C O Round Smooth Entire Opaque - A 6-9 (7) 2-20 4-40 (28)

NCCP-176 + R PY Circular Smooth Entire Opaque - A 6-9 (8) 1-35 4-45 (35)

NCCP-184 + C C Round Smooth Entire Opaque - A 5-9 (8) 2-30 10-40 (35)

NCCP-204 + C C Round Smooth Entire Opaque - A 5-9 (8) 2-30 10-40 (35)

NCCP-701 - R W Circular Smooth Entire Transparent + An 6-10 (8) 2-30 15-40 (30) NCCP-704 + C C Round Smooth Entire Opaque - A 5-9 (8) 2-30 10-40 (35)

NCCP-705 + C Orange Circular Smooth Entire Opaque - A 6-10 (8) 1-30 4-50 (37)

NCCP-706 + R C Irregular Rough Lobate Opaque + A 5-9 (7) 2-18 4-45 (28)

NCCP-708 - R Cream Circular Smooth Entire Opaque + A 5-9(8) 0-30 4-45 (37)

NCCP-710 + C Orange Circular Smooth Entire Opaque - A 6-10 (8) 1-30 4-50 (37)

NCCP-713 - R Cream Circular Smooth Entire Opaque A 6-10 (8) 0-25 4-45 (30)

NCCP-716 + R Cream Circular Rough Irregular Opaque + A 5-10 (7) 1-25 4-50(37)

NCCP-717 + R PY Circular Shiny Entire Opaque - An 6-9 (8) 1-30 4-40 (30) NCCP-718 + C Orange Circular Smooth Entire Opaque - A 6-9 (7) 0-25 15-45 (35)

NCCP-721 + R PY Circular Smooth Entire Opaque + A 8-10 (9) 0-25 10-45(37)

NCCP-722 + C C Round Smooth Entire Opaque - A 5-9 (8) 2-30 10-40 (35)

NCCP-724 + R PY Circular Smooth Entire Opaque + A 6-9 (7) 2-35 4-45 (30)

NCCP-726 W Irregular Smooth Irregular Opaque A 6-10 (7) 0-20 4-35 (28)

NCCP-727 W Irregular Smooth Irregular Opaque A 6-10 (7) 0-20 4-35 (28)

NCCP-729 + R W Round Mucoid Entire Transparent - A 6-10 (7) 2-20 10-45 (30)

NCCP-730 + C C Round Smooth Entire Opaque - A 5-9 (8) 2-30 10-40 (35) NCCP-731 + R Cream Circular Rough Irregular Opaque + A 5-10 (7) 1-25 4-50 (37)

NCCP-732 + C Orange Circular Smooth Entire Opaque - A 6-10 (8) 1-30 4-50 (37)

NCCP-734 + C Orange Circular Smooth Entire Opaque - A 6-10 (8) 1-30 4-50 (37)

NCCP-735 - R W Circular Smooth Entire Opaque - A 6-9 (8) 3-35 4-45 (37)

Roohi et al., / Int. J. Agric. Biol., Vol. 16, No. 3, 2014

568

basis of tolerance to different NaCl concentrations into

slightly, moderately, and extremely-halophilic bacteria.

However, this approach is practically ineffective for this

purpose as the optimum and range of halophilic bacteria for

tolerating NaCl is critical. Previously, the halophilic bacteria

were grouped by using the results of salt tolerance test

proposed by Kushner (1993). Our results showed that the

slightly- and moderately halophilic bacteria were more

abundant than the extremely halophilic bacteria (Table 2).

Molecular phylogeny provides information regarding

organism relationships that is the basis for the conventional

identification techniques (Singh et al., 2007). 16S rRNA

gene sequence analysis is a widely used tool for

reconstructing microbial phylogeny (Dewhirst et al., 2005).

Table 3: Molecular identification of halophilic/halotolerant bacterial strains

Strain ID Strain Name /

Genus

16S rRNA

gene (nt)

Accession Closely Related validly published taxa Similarity of 16S

rRNA gene sequence (%)

No. of Species having

>97% similarity of 16S rRNA gene

NCCP-59 Bacillus sp. 1435 AB698777 Bacillus sonorensis (AF302118) 99.36 14 NCCP-60 Planococcus sp. 1441 AB698778 Planococcus citreus (X62172) 98.39 5

NCCP-61 Bacillus sp. 1452 AB715332 Bacillus subtilissubsp. inaquosorum, (EU138467) 99.74 15

NCCP-62 Bacillus sp. 1541 AB698779 Bacillus aquimaris (AF483625) 98.17 3

NCCP-63 Bacillus sp. 1499 AB698780 Bacillus licheniformis (AE017333) 99.60 10

NCCP-65 Bacillus sp. 1132 AB715333 Bacillus safensis(AF234854) 100.00 9

NCCP-66 Jeotgalicoccus sp. 1137 AB735682 Jeotgalicoccus halotolerans (AY028925) 99.82 6

NCCP-71 Bacillus sp. 1547 AB575949 Bacillus clausii (X76440) 99.18 2

NCCP-73 Bacillus sp. 1452 AB715334 Bacillus safensis (AF234854) 98.46 5 NCCP-74 Bacillus sp. 1096 AB715335 Bacillus safensis (AF234854) 99.90 5

NCCP-75 Bacillus sp. 1174 AB715336 Bacillus safensis (AF234854) 98.86 5

NCCP-77 Bacillus sp. 1410 AB698788 Bacillus subtilissubsp. Inaquosorum (EU138467) 99.91 12

NCCP-79 Bacillus sp. 1423 AB698789 Bacillus oceanisediminis (GQ292772) 98.85 3

NCCP-80 Bacillus sp. 1128 AB715337 Bacillus methylotrophicus (EU194897) 99.73 15

NCCP-81 Bacillus sp. 1455 AB715338 Bacillus licheniformis (AE017333) 98.76 10

NCCP-82 Bacillus sp. 1480 AB715339 Bacillus clausii (X76440) 99.24 2 NCCP-83 Bacillus sp. 1125 AB715340 Bacillus safensis (AF234854) 99.08 5

NCCP-84 Staphylococcus sp. 1101 AB715341 Staphylococcus equorum subsp. linens (AF527483) 99.54 15

NCCP-85 Bacillus sp. 1169 AB715342 Bacillus safensis (AF234854) 99.91 5

NCCP-86 Thalasobacillus sp. 1434 AB698790 Thalassobacillus devorans (AJ717299) 98.95 4

NCCP-87 Halomonas sp. 790 AB735683 Halomonas boliviensis (AY245449) 99.49 6

NCCP-88 Bacillus sp. 967 AB715343 Bacillus safensis (AF234854) 99.69 5

NCCP-91 Bacillus sp. 1421 AB698793 Bacillus vietnamensis (AB099708) 98.45 3

NCCP-92 Brevibacterium sp. 1406 AB698794 Brevibacteriumcasei (X76564) 99.10 8 NCCP-93 Bacillus sp. 1418 AB698795 Bacillus endophyticus (AF295302) 99.50 1

NCCP-166 Bacillus sp. 1432 AB698798 Bacillus clausii (X76440) 98.81 2

NCCP-168 Bacillus sp. 1410 AB618147 Bacillus seohaeanensis (AY667495) 97.06 1

NCCP-170 Gracilibacillus sp. 1453 AB698801 Gracilibacillus dipsosauri (X82436) 98.81 2

NCCP-171 Halomonas sp. 1427 AB698802 Halomonas elongate (FN869568) 99.43 6

NCCP-172 Bacillus sp. 1423 AB698803 Bacillus safensis (AF234854) 99.86 5

NCCP-173 Bacillus sp. 1422 AB698804 Bacillus safensis (AF234854) 99.93 5

NCCP-174 Bacillus sp. 1433 AB698805 Bacillus licheniformis (AE017333) 98.32 14 NCCP-175 Kocuria sp. 1404 AB698806 Kocuria sediminis (JF896464) 99.78 9

NCCP-176 Halobacillus sp. 1480 AB698807 Halobacillus profundi (AB189298) 95.00 0

NCCP-184 Staphylococcus sp. 1050 AB735684 Staphylococcus equorum subsp. equorum(AB009939) 99.90 23

NCCP-204 Staphylococcus sp. 1420 AB698815 Staphylococcus equorum subsp. equorum(AB009939) 98.44 10

NCCP-701 Salinivibrio sp. 1459 AB715344 Salinivibrio sharmensis (AM279734) 97.82 1

NCCP-704 Staphylococcus sp. 1147 AB715345 Staphylococcus equorum subsp. equorum(AB009939) 100.0 23

NCCP-705 Salinicoccus sp. 1175 AB715346 Salinicoccus roseus (X94559) 98.97 4 NCCP-706 Bacillus sp. 1042 AB715347 Bacillus sonorensis (AF302118) 98.46 13

NCCP-708 Salinivibrio sp. 1100 AB735685 Salinivibrio costicola subsp. Alkaliphilus (AJ640132) 100.00 6

NCCP-710 Salinicoccus sp. 1311 Salinicoccus roseus (X94559) 98.62 3

NCCP-713 Halomonas sp. 868 AB735686 Halomonasalk aliantarctica (AJ564880) 99.42 9

NCCP-716 Bacillus sp. 1172 AB735687 Bacillus safensis (AF234854) 100.0 6

NCCP-717 Jeotgalicoccus sp. 1201 AB735688 Jeotgalicoccus halotolerans (AY028925) 99.42 6

NCCP-718 Kocuria sp. 1055 AB735689 Kocuria rosea (X87756) 99.53 7

NCCP-721 Oceanobacillus sp. 985 AB735690 Oceanobacillus onchorhynchi subsp. Incaldanensid (AJ640134) 99.90 4 NCCP-722 Staphylococcus sp. 1097 AB735691 Staphylococcus equorum subsp. equorum(AB009939) 100.0 21

NCCP-724 Oceanobacillus sp. 1172 AB735692 Oceanobacillus picturae (AJ315060) 100.0 4

NCCP-726 Bacillus sp. 1081 AB735693 Bacillus aerophilus (AJ831844) 99.54 5

NCCP-727 Bacillus sp. 1117 AB735694 Bacillus aerophilus (AJ831844) 100.0 5

NCCP-729 Bacillus sp. 966 AB735695 Bacillus endophyticus (AF295302) 99.79 1

NCCP-730 Staphylococcus sp. 1095 AB735696 Staphylococcus equorum subsp. Equorum (AB009939) 99.91 18

NCCP-731 Bacillus sp. 1134 AB735697 Bacillus safensis (AF234854) 99.65 5

NCCP-732 Bacillus sp. 1096 AB735698 Bacillus sonorensis (AF302118) 99.18 16 NCCP-734 Bacillus sp. 1176 AB735699 Bacillus sonorensis(AF302118) 99.23 16

NCCP-735 Halomonas sp. 1149 AB735700 Halomonas alkaliphila (AJ640133) 99.91 14

Diversity of Halotolerant/Halophilic Bacteria / Int. J. Agric. Biol., Vol. 16, No. 3, 2014

569

In a previous study, Rohban et al. (2009) also isolated

bacterial strains belonging to the genera Salicola,

Halovibrio, Halomonas, Bacillus, Oceanobacillus,

Thalassobacillus, Virgibacillus, Gracilibacillus,

Halobacillus, Piscibacillus and Salinicoccus from Saltan

lake of Iran.

Staphylococci has ability to grow in a wide range of

salt concentrations (Graham and Wilkinson, 1992; Garzoni

and Kelley, 2009; Morikawa et al., 2010) and is in

accordance with our study. Staphylococcus strains were

isolated from various salt samples during these studies and

the results agreed with previous reports. Highly diverse

nature of Bacillus genus found in this study is in accordance

with the observations made by Claus and Berkeley (1986).

Due to the ubiquity and capability to survive under adverse

conditions, heterotrophic Bacillus strains cannot be

considered species of certain specific habitats (Claus and

Berkeley, 1986). It is therefore not surprising that a large

number of the isolates recovered in the present study

belonged to the same bacterial group.

In conclusion, the strains from salt mines of Karak can

be further characterized using polyphasic taxonomic

approach and other molecular methods. This is the first

study on investigating the bacterial diversity of the Karak

salt mines of Pakistan and it provided a large number of

strains which are candidate novel species. The rich diversity

of genus Bacillus found in this study points out to the

extensive distribution and ecological relationship with other

microorganisms, e.g. halophilic bacteria demands further

comprehensive study. The microbial diversity can prove to

be a valuable future resource in various industrial and

biotechnological processes. Such microbes can also be used

as a source of gene(s) that can increase salt tolerance in

different crop species through genetic transformation.

Acknowledgements

This work was supported partly by financial assistance from

PSDP funded Project Research for Agricultural

Development Project under a sub-project (Grant No. CS-

55/RADP/PARC) entitled “Establishment of Microbial Bio-

Resource Laboratories: National Culture Collection of

Pakistan (NCCP)” from Pakistan Agricultural Research

Council, Islamabad, Pakistan

References

Ahmed, I., A. Yokota and T. Fujiwara, 2007. A novel highly boron tolerant

bacterium, bacillus boroniphilus sp. Nov., isolated from soil, that

requires boron for its growth. Extremophiles, 11: 217‒224

Antón, J., R. Rosselló‒Mora, F. Rodríguez‒Valera and R. Amann, 2000.

Extremely halophilic bacteria in crystallizer ponds from solar

salterns. Appl. Environ. Microbiol., 66: 3052‒3057

Birbir, M., B. Calli, B. Mertoglu, R. Bardavid, A. Oren, M. Ogmen and A.

Ogan, 2007. Extremely halophilic archaea from tuz lake, turkey, and

the adjacent kaldirim and kayacik salterns. World J. Microbiol.

Biotechnol. 23: 309‒316

Castillo, A.M., M.C. Gutiérrez, M. Kamekura, Y. Ma, D.A. Cowan, B.E.

Jones, W.D. Grant and A. Ventosa, 2006. Halovivax asiaticus gen.

Nov., sp. Nov., a novel extremely halophilic archaeon isolated

from inner mongolia, china. Int. J. Syst. Evol. Microbiol., 56: 765‒770

Castillo, A.M., M.C. Gutiérrez, M. Kamekura, Y. Xue, Y. Ma, D.A. Cowan,

B.E. Jones, W.D. Grant and A. Ventosa, 2007. Halorubrum

ejinorense sp. Nov., isolated from lake ejinor, inner mongolia, china.

Int. J. Syst. Evol. Microbiol., 57: 2538‒2542

Fig. 2: Phylogenetic tree showing the interrelationships of

isolated halotolerant/halophilic strains along with their

closely related taxa belonging to Firmicutes, Actinobacteria

and Proteobacteria inferred from 16SrRNA gene sequence.

Data with gaps were removed after alignment by

CLUXTAL X. The rooted tree was constructed using the

neighbour-joining method contained in the MEGA5

software (Tamura et al., 2011). Bootstrap values, expressed

as percentages of 1000 replications, are given at branching

points. Closely related type species were presented in bold

letters and the 3% bar shows sequence divergence

Roohi et al., / Int. J. Agric. Biol., Vol. 16, No. 3, 2014

570

Claus, D. and R.C.W. Berkeley, 1986. Genus Bacillus 1872. In: Bergey's

Manual of Systematic Bacteriology. P.H.A. Sneath, PHA, N.S. Mair,

M.E. Sharpe and J.G. Holt (ed.). Vol. 2. pp: 1105‒1139. Williams

and Wilkins, Baltimore, Maryland, USA

Dewhirst, F.E., Z. Shen, M.S. Scimeca, L.N. Stokes, T. Boumenna, T.

Chen, B.J. Paster and J.G. Fox, 2005. Discordant 16s and 23s rrna

gene phylogenies for the genus helicobacter: implications for

phylogenetic inference and systematics. J. Bacteriol., 187: 6106‒6118

Euzéby, J.P., 2013. List of prokaryotic names with standing in

nomenclature. Accessed on 9 June 2013 http:

//www.bacterio.net/h/halobacteriaceae.html

Fritze, D., 2002. Bacillus identification‒ traditional approaches. In:

Applications and Systematics of Bacillus and Relatives. Berkeley, R.,

M. Heyndrickx, N. Logan, P. De Vos (Eds.), pp. 100‒122.

Blackwell, Oxford, UK

Garzoni, C. and W.L. Kelley, 2009. Staphylococcus aureus: New evidence

for intracellular persistence. Trends Microbiol., 17: 59‒65

Graham, J.E. and B.J. Wilkinson, 1992. Staphylococcus aureus

osmoregulation: roles for choline, glycine betaine, proline, and

taurine. J. Bacteriol., 174: 2711‒2716

Hall, T.A., 1999. Bioedit: a user‒friendly biological sequence alignment

editor and analysis program for windows 95/98/nt. Nucleic Acids

Symposium Series

Johnson, A., L. Thurlow, R. Zwenger and E. Gillock, 2007. Partial

characterization of two moderately halophilic bacteria from a kansas

salt marsh. Prairie Nat, 39: 29‒39

Kamekura, M., 1998. Diversity of extremely halophilic bacteria.

Extremophiles, 2: 289‒295

Kushner, D.J., 1985. The halobacteriaceae. Academic Press, New York,

USA

Kushner, D.J., 1993. Growth and nutrition of halophilic bacteria. CRC

Press, Boca Raton, Florida, USA

Litchfield, C.D. and P.M. Gillevet, 2002. Microbial diversity and

complexity in hypersaline environments: a preliminary assessment.

J. Ind. Microbiol. Biotechnol., 28: 48‒55

Mancinelli, R.L. and L.I. Hochstein, 1986. The occurrence of denitrification

in extremely halophilic bacteria. FEMS Microbiol. Lett., 35: 55‒58

Morikawa, K., R.L. Ohniwa, T. Ohta, Y. Tanaka, K. Takeyasu and T.

Msadek, 2010. Adaptation beyond the stress response: Cell structure

dynamics and population heterogeneity in staphylococcus aureus.

Microb. Environ., 25: 75‒82

Oren, A., 1993. Ecology of extremely halophilic microorganisms. CRC

Press, Boca Raton, Florida, USA

Oren, A., 2002. Diversity of halophilic microorganisms: environments,

phylogeny, physiology, and applications. J. Ind. Microbiol.

Biotechnol., 28: 56‒63

Oren, A. and F.s. Rodrı́guez‒Valera, 2001. The contribution of halophilic

bacteria to the red coloration of saltern crystallizer ponds1. FEMS

Microbiol. Ecol., 36: 123‒130

Oren, A., A. Ventosa and W.D. Grant, 1997. Proposed minimal standards

for description of new taxa in the order halobacteriales. Int. J. Syst.

Bacteriol., 47: 233‒238

Ozcan, B., G. Ozcengiz, A. Coleri and C. Cokmus, 2007. Diversity of

halophilic archaea from six distinct parts of turkey. J. Microbiol.

Biotechnol., 17: 985–992

PMDC, 2013. Pakistan mineral development corporation. Ministry of

Petroleum and Natural Resources, Government of Pakistan,

Islamabad

Quesada, E., V. Béjar, M. Ferrer, C. Calvo, I. Llamas, F. Martínez‒Checa,

S. Arias, C. Ruiz‒García, R. Páez, M. Martinez‒Cánovas and A. del

Moral, 2004. Moderately halophilic, exopolysaccharide‒producing

bacteria. Berlin: Springer‒Verlag, Berlin, Germany

Quesada, E., A. Ventosa, F. Rodriguez‒Valera and A. Ramos‒Cormenzana,

1982. Types and properties of some bacteria isolated from

hypersaline soils. J. Appl. Microbiol., 53: 155‒161

Rohban, R., M. Amoozegar and A. Ventosa, 2009. Screening and isolation

of halophilic bacteria producing extracellular hydrolyses from howz

soltan lake, Iran. J. Ind. Microbiol. Biotechnol., 36: 333‒340

Roohi, A., I. Ahmed, M. Iqbal and M. Jamil, 2012. Preliminary isolation

and characterization of halotolerant and halophilic bacteria from salt

mines of Karak, Pakistan. Pak. J. Bot., 44: 365‒370

Singh, S., R. Chandra, D.K. Patel and V. Rai, 2007. Isolation and

characterization of novel serratia marcescens (ay927692) for

pentachlorophenol degradation from pulp and paper mill waste.

World J. Microbiol. Biotechnol.,23: 1747‒1754

Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei and S. Kumar,

2011. Mega5: Molecular evolutionary genetics analysis using

maximum likelihood, evolutionary distance, and maximum

parsimony methods. Mol. Biol. Evol., 28: 2731‒2739

Ventosa, A., E. Mellado, C. Sánchez‒Porro and M. Marquez , 2008.

Halophilic and halotolerant micro‒organisms from soils. Berlin

Springer‒Verlag, Berlin, Germany

Woese, C.R. and G.E. Fox, 1977. Phylogenetic structure of the prokaryotic

domain: The primary kingdoms. Proc. Natl. Acad. Sci. USA, 74:

5088‒5090

(Received 30 September 2013; Accepted 24 February 2014)